A decade ago, gene expression seemed so straightforward: genes were
either switched on or off. Not both. Then in 2006, a blockbuster finding
reported that developmentally regulated genes in mouse embryonic stem
cells can have marks associated with both active and repressed genes,
and that such genes, which were referred to as "bivalently marked
genes," can be committed to one way or another during development and
differentiation. This paradoxical state -- akin to figuring out how to
navigate a red and green traffic signal -- has since undergone scrutiny
by labs worldwide. What has been postulated is that the control regions
(or promoters) of some genes, particularly those critical for
development during the undifferentiated state, stay "poised" for
plasticity by communicating with both activating and repressive
histones, a state biologists term "bivalency."

A study by researchers at the Stowers Institute for Medical Research
now revisits that notion. In this week's advance online edition of the
journal Nature Structural and Molecular Biology, a team led by
Investigator Ali Shilatifard, Ph.D., identifies the protein complex that
implements the activating histone mark specifically at "poised" genes
in mouse embryonic stem (ES) cells, but reports that its loss has little
effect on developmental gene activation during differentiation. This
suggests that there is more to learn about interpreting histone
modification patterns in embryonic and even cancer cells.

"There has been a lot of excitement over the idea that promoters of
developmentally regulated genes exhibit both the stop and go signals,"
explains Shilatifard. "That work supports the idea that histone
modifications could constitute a code that regulates gene expression.
However, we have argued that the code is not absolute and is context
dependent."

Shilatifard has a historic interest in gene regulation governing
development and cancer. In 2001, his laboratory was the first to
characterize a complex of yeast proteins called COMPASS, which
enzymatically methylates histones in a way that favors gene expression.
Later, he discovered that mammals have six COMPASS look-alikes -- two
SET proteins (1A and 1B) and four MLL (Mixed-Lineage Leukemia) proteins,
the latter so named because they are mutant in some leukemias. The
group has since focused on understanding functional differences among
the COMPASS methylases. The role of mouse Mll2 in establishing bivalency
was the topic of the latest study.

Comprehending the results of the paper requires a brief primer
defining three potential methylation states of histone H3. If the 4th
amino acid, lysine (K), displays three methyl groups (designated
H3K4me3), then this mark is a sign of active transcription from that
region of the chromosome. If the 27th residue of histone H3 (also a
lysine) is trimethylated (H3K27me3), this mark is associated with the
silencing of that region of the chromosome. But if both histone H3
residues are marked by methylation (H3K4me3 and H3K27me3 marks), that
gene is deemed poised for activation in the undifferentiated cell state.

The team already knew that an enzyme complex called PRC2 implemented
the repressive H3K27me3 mark. To identify which COMPASS family member is
involved in this process, the group genetically eliminated all
possibilities and came up with Mll2 as the responsible factor.
Mll2-deficient cells indeed show H3K4me3 loss, not at all genes, but at
the promoters of developmentally regulated genes, such as the Hox genes.
The revelation came when the researchers evaluated behaviors of
Mll2-deficient mouse embryonic stem cells. First, the cells continued to
display the defining property of a stem cell, the ability to
"self-renew," meaning that genes that permit stem cell versatility were
undisturbed by Mll2 loss. But remarkably, when cultured with a factor
that induces their maturation, Mll2-deficient mouse ES cells showed no
apparent abnormalities in gene expression. In fact, expression of the
very Hox genes that normally exhibit bivalent histone marks was as timely in Mll2-deficient cells as it was in non-mutant cells.

"This means that Mll2-deficient mouse ES cells that receive a
differentiation signal can still activate genes required for maturation,
even though they have lost the H3K4me3 mark on bivalent regions" says
Deqing Hu, Ph.D., the postdoctoral fellow who led the study. "This work
paves the way for understanding what the real function of bivalency is
in pluripotent cells and development."

The study's findings also potentially impact oncogenesis, as
tumor-initiating "cancer stem cells" exhibit bivalent histone marks at
some genes. "Cancer stem cells are resistant to chemotherapy, making
them difficult to eradicate," says Hu. "Our work could shed light on how
cancer stem cells form a tumor or suggest a way to shut these genes
down."

Other Stowers contributors from the Shilatifard lab were Alexander S.
Garruss, Xin Gao, Marc A Morgan, Ph.D., Malcolm Cook, and Edwin R
Smith, Ph.D.
Funding for the study came from the Stowers Institute for Medical Research and the United States National Cancer Institute.